Advancing Circulating Tumor Cell Isolation and Analysis: Development of Optimized Immunomagnetic Techniques and Molecular Characterization Approaches for Early Cancer Detection
Peng Liu is a PhD student in the Department of Medical Cell Biophysics.(Co)Promotors are prof.dr. L.W.M.M. Terstappen and prof.dr.ir. P. Jonkheijm from the Faculty of Science & Technology.
Circulating tumor cells (CTCs) play a pivotal role in cancer metastasis and offer significant potential for improving cancer diagnostics and personalized therapies. This thesis focuses on overcoming the challenges associated with the isolation and analysis of CTCs by developing innovative methodologies that enhance capture efficiency, address tumor heterogeneity, and preserve molecular integrity for downstream analyses.
Recognizing the limitations of existing CTC isolation methods—particularly the low antigen expression on CTCs and tumor heterogeneity—the research undertakes a multifaceted approach to address these challenges. In the initial phase, a thorough literature review is conducted to elucidate the shortcomings of current CTC isolation techniques, including physical property-based methods (e.g., filtration, density gradient centrifugation) and biological property-based approaches that rely heavily on the epithelial cell adhesion molecule (EpCAM). These methods often fail to capture the full spectrum of CTCs due to their heterogeneous nature and variable EpCAM expression, emphasizing the necessity for more effective strategies (chapter 1).
To identify the optimal magnetic beads for enhancing CTC isolation, the thesis evaluates eight commercially available streptavidin-coated magnetic beads using a flow-through immunomagnetic enrichment system (chapter 2). By systematically comparing their performance in capturing EpCAM-expressing cancer cell lines, the study determines the bead size and properties that maximize capture efficiency while minimizing nonspecific binding. The findings reveal that beads in the size range of approximately 100 to 150 nanometers offer the best balance between magnetic responsiveness and specificity. However, limitations in capturing CTCs with low EpCAM expression persist, highlighting the need for custom-designed nanobeads with optimized characteristics.
Building upon these insights, the thesis presents the synthesis and detailed characterization of silica-coated magnetic nanobeads functionalized with streptavidin (NC@silica-SA) (chapter 3). By optimizing parameters such as particle size, magnetic properties, and surface chemistry, these nanobeads demonstrate enhanced magnetic responsiveness and significantly reduced nonspecific interactions with non-target cells. This advancement leads to a substantial improvement in both the efficiency and purity of CTC capture, particularly for cancer cells exhibiting low levels of EpCAM expression, thereby addressing a critical limitation of existing immunomagnetic separation methods.
Addressing tumor heterogeneity, a significant barrier to effective CTC isolation, the thesis investigates the use of recombinant VAR2CSA protein (rVAR2) (chapter 4). This protein specifically binds to oncofetal chondroitin sulfate, a molecule expressed on the surface of a wide range of cancer cells but not on normal adult cells. By combining rVAR2 with anti-EpCAM antibodies, the research achieves a dual-targeting approach that significantly enhances overall CTC capture efficiency across various non-small-cell lung cancer (NSCLC) cell lines. This strategy effectively broadens the spectrum of detectable CTCs by targeting multiple cell surface markers, thereby capturing heterogeneous CTC populations that might be missed by single-marker methods.
Finally, to preserve the molecular integrity of isolated CTCs for downstream molecular analyses, the thesis develops innovative staining and isolation protocols that eliminate the need for cell permeabilization and fixation, processes that can degrade intracellular nucleic acids (chapter 5). Utilizing rVAR2 staining in conjunction with single-cell magnetic pickup techniques, the methods maintain the integrity of mRNA within the isolated cells. This preservation is crucial for accurate molecular analyses at the single-cell level, such as reverse transcription quantitative polymerase chain reaction (RT-qPCR) and potentially single-cell RNA sequencing. These analyses facilitate a deeper understanding of tumor biology, gene expression profiles, and cellular heterogeneity, which are essential for the development of personalized therapeutic strategies.
In conclusion, this thesis makes significant contributions to the field of CTC research by developing and validating novel methodologies that enhance the detection, isolation, and characterization of CTCs. By addressing critical limitations of current methods—such as low antigen expression, tumor heterogeneity, and the preservation of molecular integrity—the work provides valuable insights with substantial implications for cancer diagnostics, prognostics, and personalized medicine. The advancements presented have the potential to improve early detection of metastasis, monitor disease progression, and inform tailored treatment decisions, ultimately contributing to better patient outcomes.
